Predictive mRNA Biomarkers for the Prediction of the Treatment with Methotrexate (MTX)

Abstract

The present invention concerns predictive mRNA biomarkers, which are used in combination with HLA-DRB4 for forecasting the treatment with MTX (methotrexate). The present invention further concerns a method for forecasting the treatment with MTX (methotrexate), comprising detection of the predictive mRNA biomarkers in combination with HLA-DRB4 in patient samples, whereby the patients are classified into responders or non-responders.

1 - 13 . (canceled) 
     
     
         14 . A method for forecasting whether a patient will be a responder or a non-responder to treatment with MTX (methotrexate), comprising the steps of
 (i) providing a patient sample,   (ii) detecting at least one mRNA biomarker selected from
 ARG1, CKAP4, CRISP3, CST3, GCLM, KIAA0564, KIAA1324, LCN2, LOC654433/PAX8-AS1, LTF, OLFM4, OSBPL1A, MMP8, SIAH1, SLC8A1/BF223010, and SULF2 
   and/or selected from
 AQP3, CFD, DEFA4, EIF5A, GATA3, Hs.674648, KCNE3, PAM, PRDX5, RNASE2, TCN1, TKT, SLC35E2, SNHG5, SPAG9, and WLS in combination with HLA-DRB4 
   in the patient sample,   and   (iii) assaying the relative expression level of the at least one mRNA biomarker and of
 HLA-DRB4 by comparison with one or more reference standards and/or control samples, 
   
       wherein the patient is classified as a responder or non-responder based on said relative expression level. 
     
     
         15 . The method as claimed in  claim 14 , wherein the treatment with MTX comprises a combination of biologics and MTX. 
     
     
         16 . The method as claimed in  claim 14 , wherein the classification of the patient is carried out prior to commencing treatment with MTX (methotrexate). 
     
     
         17 . The method as claimed in  claim 14 , wherein the sample is pre-selected into either a HLA-DRB4-positive or a HLA-DRB4-negative sample. 
     
     
         18 . The method as claimed in  claim 14 , wherein the sample undergoes a pre-treatment comprising removing globin mRNA, reverse transcription of the total mRNA and/or labelling with a label. 
     
     
         19 . The method as claimed in  claim 14 , wherein the detection in step (ii) comprises assaying for the presence of the mRNA marker and its expression levels,
 or comprises assaying for the presence of an miRNA marker.   
     
     
         20 . The method as claimed in  claim 19 , wherein the assay is carried out using
 serial analysis of gene expression (SAGE), real time quantitative PCR (qPCR), bead technology, blot, RNA or next-generation sequencing, and/or   in-situ hybridization, Northern blot, DNA microarrays or DNA macroarrays.   
     
     
         21 . The method as claimed in  claim 14 , wherein the patient to be treated has an inflammatory disease, chronic inflammatory disease, autoimmune disease and/or tumor disease. 
     
     
         22 . The method as claimed in  claim 21 , wherein the inflammatory, chronic inflammatory, or autoimmune disease is
 selected from rheumatoid arthritis (RA), primary chronic polyarthritis, juvenile idiopathic arthritis, systemic lupus erythematosus (SLE), systemic sclerosis (scleroderma), polymyositis, dermatomyositis, inclusion body myositis, psoriasis, multiple sclerosis, uveitis, Crohn's disease, Churg-Strauss syndrome (CSS), Boeck's disease, Bechterew's disease, relapsing polychondritis, colitis ulcerosa, polymyalgia rheumatica, giant cell arteritis, vasculitis, and myositides; and   the tumor disease is selected from: acute lymphatic leukemia (ALL) (juvenile and adult), transitional cell carcinoma of the bladder, breast cancer, medulloblastoma, ependymoma (juvenile and adult), non-Hodgkins lymphoma (NHL) (juvenile and adult), and osteosarcoma (juvenile and adult).   
     
     
         23 . The method as claimed in  claim 14 , wherein at least 50% of the mRNA biomarker genes are detected in combination with HLA-DRB4. 
     
     
         24 . The method as claimed in  claim 14 , wherein in the case of treatment of rheumatoid arthritis (RA), all 32 biomarker genes (100%) are assayed in combination with HLA-DRB4. 
     
     
         25 . The method as claimed in  claim 14 , wherein the sample is selected from whole blood, peripheral blood leukocytes and purified blood cells. 
     
     
         26 . The method as claimed in  claim 14 , wherein in step (ii), at least one mRNA biomarker(s) selected from
 CKAP4, CRISP3, KIAA0564, LCN2, OLFM4, MMP8, and SLC8A1/BF223010   and/or selected from   AQP3, DEFA4, or and SNHG5 is detected in combination with HLA-DRB4 in the patient sample.   
     
     
         27 . The method as claimed in  claim 14 , wherein in step (iii), standard samples containing one or more housekeeping genes and control samples are samples of responders and/or non-responders. 
     
     
         28 . A kit for forecasting whether a patient will be a responder or a non-responder to treatment with MTX (methotrexate), comprising
 (a) implementing means for detecting at least one mRNA biomarker selected from
 ARG1, CKAP4, CRISP3, CST3, GCLM, KIAA0564, KIAA1324, LCN2, LOC654433/PAX8-AS1, LTF, OLFM4, OSBPL1A, MMP8, SIAH1, SLC8A1/BF223010, and SULF2 
   and/or selected from
 AQP3, CFD, DEFA4, EIF5A, GATA3, Hs.674648, KCNE3, PAM, PRDX5, RNASE2, TCN1, TKT, SLC35E2, SNHG5, SPAG9, and WLS 
 in combination with HLA-DRB4 
 in a patient sample, 
   (b) at least one reference standard comprising a sample containing one or more housekeeping genes, and   (c) at least one control sample comprising a sample from a responder and/or a non-responder.   
     
     
         29 . The kit as claimed in  claim 28 , wherein the implementing means for detecting at least one mRNA biomarker detects a biomarker selected from
 CKAP4, CRISP3, KIAA0564, LCN2, OLFM4, MMP8, and SLC8A1/BF223010   and/or selected from   AQP3, DEFA4, and SNHG5.   
     
     
         30 . The kit as claimed in  claim 28 , wherein the implementing means for detecting at least one mRNA biomarker in a patient sample comprises:
 arrays, chips,   primers,   marker and labels.